Computational and Experimental Studies of Structure/ Dynamics of HDL Assemblies

HDL 组件结构/动力学的计算和实验研究

• 批准号: 8242746
• 负责人: Jere P Segrest
• 金额: $ 23.96万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8242746
• 关键词: Apolipoproteins; Apolipoproteins A; Atherosclerosis; Chemicals; Cholesterol; Cholesterol Esters; Complex; Coronary heart disease; Cryoelectron Microscopy; Dimerization; Enzymes; Funding; Goals; High Density Lipoprotein Cholesterol; High Density Lipoproteins; Image; In Vitro; Investigation; Kinetics; Lead; Length; Lipids; Lipoproteins; Maps; Membrane; Metabolic; Modeling; Molecular; Molecular Models; Mutation; Pathway interactions; Pattern; Peptides; Pharmaceutical Preparations; Phosphatidylcholine-Sterol O-Acyltransferase; Phospholipids; Plasma; Positioning Attribute; Property; Proteins; Pump; Registries; Role; Series; Sodium Chloride; Sphingomyelins; Structure; Surface; Testing; absorption; base; computer studies; design; dimer; flexibility; insight; mimetics; molecular dynamics; molecular modeling; particle; prevent; reconstitution; research study; simulation; stoichiometry

The goals of this project are to continue our computational and experimental investigations of HDL, focusing on mechanisms of assembly of phospholipid (PL)-rich and cholesteryl ester (CE)-rich HDL from PL-poor apoA-l. These goals are driven by our lead hypothesis that apoA-l is a uniquely elastic bilayer-bounding protein capable of absorbing PL and CE in small molecular increments. To achieve these goals, we propose, using a combination of molecular dynamics (MD) and experimental approaches, two specific aims listed in order of priority: 1) To study the structure/dynamics of PL-rich HDL. To achieve this aim, we will: i) Perform more robust simulations of the 100:2 and 50:2 particles, ii) Study the role of rotamer registry by varying registry and inhibiting buried salt bridges via MD simulations and experimentally via dimerization of Cys mutations, iii) Determine the role of the flexible domain of full length apoA-l and the effect of unesterified cholesterol (UC) and/or sphingomyelin (SM) via simulations and experimental studies, the role of salt bridge formation between lipid headgroups and apoA-l, and the mechanisms of interaction of peptide mimetics with PL-rich HDL via simulations, iv) Image PL-rich HDL assemblies using cryoEM and low angle x-ray scattering 2) To explore the molecular basis for the assembly of PL-rich HDL from PL-poor apoA-l. To achieve this aim, we will use: i) MD simulations of monomeric apoA-l from a recent x-ray crystal structure, and dimeric PLD-poor HDL using the particle shrinkage approach; ii) experimental approaches to: determine stoichiometry and kinetics of assembly of PL-rich from PL-poor HDL, determine composition/stoichiometry of pre-beta (PL-poor) HDL from plasma, and determine the structure/composition of PL-rich HDL produced by the ABCA1 pathway. Preliminary results suggest that the majority of these aims can be achieved within the 5 years of requested support. A long-term aim for which no funds are requested is: To determine the structure/dynamics of CE-rich HDL. Because of the extraordinary power of MD simulation to provide supramolecular images of HDL as indicated by our preliminary results, by combining MD simulations and experimental approaches, we are uniquely positioned to gain new insights into the structure/function of the various HDL subspecies. As a long term goal, from the resulting dynamic structure-function maps, drugs will be designed to modify the "good" cholesterol, HDL, to prevent or reverse coronary heart disease.

本项目的目标是继续我们对高密度脂蛋白的计算和实验研究，重点是从缺乏磷脂的载脂蛋白A-L中组装富含磷脂(PL)和富含胆固醇酯(CE)的高密度脂蛋白的机制。这些目标是由我们的领先假设驱动的，即载脂蛋白A-L是一种独特的弹性双层结合蛋白，能够以小分子增量吸收PL和CE。为了实现这些目标，我们结合分子动力学(MD)和实验方法，提出了两个具体的目标，按优先顺序列出：1)研究富含PL的高密度脂蛋白的结构/动力学。为了实现这一目标，我们将：i)执行 对100：2和50：2粒子进行更稳健的模拟，ii)研究旋转异构体的作用，通过分子动力学模拟和半胱氨酸突变的二聚化来改变注册和抑制隐藏的盐桥，iii)通过模拟和实验研究确定全长apoA-L的柔性结构域的作用以及未酯化胆固醇(UC)和/或鞘磷脂(SM)的影响，脂头基团和apoA-L之间的盐桥形成的作用，以及多肽模拟物与富含PL的高密度脂蛋白相互作用的机制 2)探讨低载脂蛋白A-L细胞组装高密度脂蛋白的分子基础。为了实现这一目标，我们将使用：i)根据最近的X射线晶体结构对单体apoA-L进行MD模拟，并使用粒子收缩方法对低PL-HDL二聚体进行MD模拟；ii)实验方法：确定由贫PL高密度脂蛋白制备的富PL高密度脂蛋白的化学计量和组装动力学，确定来自血浆的前β(贫PL)高密度脂蛋白的组成/化学计量比，以及确定ABCA1途径产生的富PL高密度脂蛋白的结构/组成。初步结果表明，这些目标中的大多数可以在请求支助的5年内实现。一个不需要资金的长期目标是：确定富含CE的高密度脂蛋白的结构/动力学。正如我们的初步结果所表明的那样，由于MD模拟提供高密度脂蛋白的超分子图像的非凡能力，通过将MD模拟与实验方法相结合，我们处于独特的地位，可以获得对各种高密度脂蛋白亚种的结构/功能的新见解。作为一个长期目标，从得到的动态结构-功能图中，药物将被设计成修改“好”的胆固醇，高密度脂蛋白，以预防或逆转冠心病。